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Lecture 40&41 Edible Vaccines Specific vaccines have been produced in plants as a result of transient or stable expression of foreign genes. It has recently been shown that genes encoding antigens of bacterial and viral pathogens can be expressed in plants in a form in which they retain native immunogenic properties. There are basically 2 strategies for edible vaccine production. These include: 1. Expression of foreign antigens in plant via stable transformation 2. Delivery of vaccine epitopes via plants virus (Mason and Arntzen, 1995) Figure 4 Strategies for the production of candidate vaccine antigens in plant tissues (Mason and Arntzen, 1995) 1 The first report of the concept of using a plant expression system for the production of an edible vaccine appeared in a patent application published under the international patent co-operatin treaty. It described a means to express a surface protein (spa A) from Streptococus mutans in tobacco plants to a level of 0.02% of total leaf protein, the gene had been stably inserted by Agrobacterium mediated transformation. Data were presented on the oral immuno genicity of spa A produced in E.coli, which stimulated the production of S-IgA in saliva (Bergmann and Waldman, 1998). This paved the way of the concept of using plants as bioreactors for obtaining edible vaccines against bacterial and viral diseases. The use of strong and organ specific promoters help for targeting the proteins in the specific organs. This helps in the stability of proteins (antigens). These antigens stimulate serum as well as mucosal immune system to produce antibody against the orally injected antigens. Thus an antigen produced in edible part act as vaccines. Till now 5 antigens have been experimented in plants: Rabies virus G protein in Tomato Norwalk virus (cold virus) capsid protein in tobacco and potato Hepatitis B surface antigens in tobacco and potato E.Coli heat labile entertoxin B subunit (LT-B) in tobacco Carbohydrateslera toxin B (CT-B) in potato 1. Hepatitis “B” surface antigens in Tobacco Hepatitis “B” virus is one of the major causes of chronic viremia in humens. Arntzen and his team (Mason et al., 1992), demonstrated that tobacco leaves can express recombinant hepatitis surface B antigen (HBsAg) to a level of 0.01% of the soluble protein content. The surface antigen (HBsAg) is produced in copious amounts in liver cells of infected individuals (Richter et al., 2000). Some of this envelope protein is secreted from liver cells can be recovered in serum in form of virus like particdes (VLPs). The VLPs, consisting of HBsAg and membrane lipids were found to be highly immunogenic and would elicit antibodies specific for authentic HBV. The gene encoding 2 HBsAg was expressed in tobacco plants, targeting the plastid (chloroplast). Expression of HBsAg in transformation tobacco leaves yield VLPs avg. 22mm in size. When used for parenteral immunization of mice, the tobacco VLPs provoked BandT cell immune response, comparable to yeast – derived vaccine. However, because of possible degradation of proteins in the gut, presentation of vaccine antigens by an oral route requires much higher levels of immunogen than parenteral delivery so by increasing the levels of HBsAg in plants tissues, it would enhance the utility of plants as source of recombinant antigen. Figure 4 Structure of expression cassettes Structures of expression cassettes 3 Figure 5 RNA Analyzed by Northern Blot Hybridised with HBs Ag coding sequence E.coli heat labile enterotoxin (B subunit )in tobacco The heat labile enterotoxin (LT) from E.coli is a multimeric protein consisting of a 27KDA A subunit (LT-A) and a pentamer of 11.6Kda B sub unit (LT-B). LT-B specifically binds to GM1, gangliosides on epithelial cells allowing entry of LT-A into cells. The gene encoding LT-B was cloned into a plant expression vector behind CaMV 35S promoter and 5’ untranslated region of tobacco etch virus (TEV). The 3’ sequence of soybean VSPB gene was used as a terminator. A second plasmid with short coding sequence leading to the endoplasmic retention was ligated to the 3’ end of LT-B coding sequence. Both the plasmids were transformed into tobacco and potato plants by Agrobacterium transformation (Haq et al., 1995). The antigen amount was found to be 0.0014% of total soluble proteins in tobacco leaves and 0.011% of microtuber proteins in potato tubers. On feeding tubers to mices, both humoral and mucosal immune responses were generated. Rabies virus “G” protein in tomato The gene for rabies virus glycoprotein including the signal peptide was linked to CaMV 35 S promoter and transformation by Agrobaterium into tomato plants. Leaves, fruits from transgenic tomato plants expressed the recombinant glycoprotein having a molecular weight of 62 Kda. The protein was detected by Western blotting and found to be localised in Golgibodies, vesicles, plasmalemma and parenchymatous cell walls (Mc Garvey et al., 1995) 4 HIV Virus The glycoprotein (gp41) of HIV type, (HIV-1, IIIb) containing 22 aminoacids was introduced between aminoacids 22 and 23 of small capsid subunit of CPMV (Porta et al., 1994). 3 different HIV-1 strains were found to be neutralized in vitro and invivo by antibodies recovered from mice previously injected with gp41 modified CPMV (McLain et al., 1995). Norwalk Virus (Cold Virus) capsid protein in tobacco and potato Norwalk virus is the causative agent of acute epidemic gastroenteritis. The Norwalk virus capsid protein (NVCP) gene was fused to CaMV 35S promoter or potato tuber specific patatin promoter using TEV translation enhancer (Mason et al., 1996). Transformation was carried out using Agrobaterium tumefaciens. Upto 0.23% of soluble protein in tobacco leaves and 0.37% of total soluble protein in potato tubers were recovered as rNVCP. 5